Use of granme b as an hsp70/hsp70 peptide dependent inducer of apoptosis in tumor cells

ABSTRACT

The present invention relates to a method of inducing or enhancing the expression of granzyme B in natural killer (NK) cells. The present invention relates also to a use of said NK cells for the preparation of a pharmaceutical composition for the treatment of tumors, viral or bacterial infections or inflammatory diseases. Further, the present invention relates to the use of granzyme B for the treatment of tumors, viral or bacterial infections or inflammatory diseases, wherein the tumor cells or the cells affected by said infection or inflammation express Hsp70 in their cell surface.

The present invention relates to a method of inducing or enhancing theexpression of granzyme B in natural killer (NK) cells. The presentinvention relates also to a use of said NK cells for the preparation ofa pharmaceutical composition for the treatment of tumors, viral orbacterial infections or inflammatory diseases. Further, the presentinvention relates to the use of granzyme B for the treatment of tumors,viral or bacterial infections or inflammatory diseases, wherein thetumor cells or the cells affected by said infection or inflammationexpress Hsp70 on their cell surface.

A variety of documents is cited throughout this specification. Thedisclosure content of said documents is herewith incorporated byreference.

Elevated cytoplasmic levels of heat shock protein 70 (Hsp70) have beenfound to protect tumor cells against programmed cell death (Nylandstedet. al. (2000) Ann. N.Y. Acad. Sci. 926, 122). Hsp70 is the major stressinducible form of the heat shock protein family (HSP), which isprimarily located in the cytosol. Evidence accumulated during recentyears has demonstrated that extracellular localized and plasmamembrane-bound HSPs are highly immunogenic and expose the cells toimmune attack (Schild et. al. (1999) Current Opinion in Immunology 11,109). Following receptor-mediated uptake (Arnold-Schild et. al. (1999)J. Immunol. 162, 3757) and re-presentation by antigen presenting cells(APC), HSP-chaperoned peptides elicit a cytotoxic, CD8⁺ T cell response(Suto et. al. (1995) Science 269, 1585). Several receptors, includingCD91 and toll-like receptors 2 and 4 (TLR2/4), have been identified thatmediate interaction of HSP90 (gp96), HSP70 (Hsp70, Hsc70) and HSP60peptide complexes with APCs (Basu et. al. (2001) Immunity 14, 303;Binder et. al. (2000) Nat. Immunol. 1, 151; Sondermann et. al. (2000)Biol. Chem. 381, 1165.; Ohashi et. al. (2000) J. Immunol. 164, 558). Apeptide-independent “chaperokine effect” has been described for membersof the HSP70 group. Binding of exogenous HSP70 to monocytes via TLR2/4in a CD14 dependent pathway induces receptor clustering and thesecretion of proinflammatory cytokines via MyD88/IRAK/NFκ-B signaltransduction (Pfeiffer et. al. (2001) Eur. J. Immunol. 31, 3153; Aseaet. al. (2000) Nature Medicine, 6, 435; Asea et. al. (2000) Cell Stress& Chaperones, 5, 425; Asea et. al. (2002) J Biol Chem. 277(17), 15028).

Natural killer (NK) cells have been found to specifically interact witha C-terminal localized epitope of Hsp70 (Botzler et. al. (1998) CellStress & Chaperones, 3, 6), that is presented on the cell membrane oftumor cells (Multhoff et. al. (1995) Int. J. Cancer, 61, 272; Multhoffet. al. (1997) J. Immunol. 158, 4341). The amount of membrane-boundHsp70 on tumor cells positively correlates with the sensitivity to thelysis mediated by NK cells: Physical (heat) as well as chemical(cytostatic drugs) stress has been found to increase Hsp70 cell surfaceexpression on tumor cells and thereby rendering them better targets forNK cells (Multhoff (1997) Int. J. Hyperthermia 13, 39; Botzler et. al.(1999) Exp. Hematol. 27, 470; Rabinovich et. al. (2000) J. Immunol. 165,2390; Feng et. al. (2001) Blood 97, 3505). Incubation of purified NKcells with recombinant Hsp70-protein increases their cytolytic activityagainst Hsp70 membrane-positive tumor cells (Multhoff et. al. (1999)Exp. Hematology 27, 1627). The same effect is achieved by a 14 aminoacid peptide, termed TKD (TKDNNLLGRFELSG, aa450-463), derived from theC-terminal domain of Hsp70. This region corresponds to the domain ofHsp70 exposed to the extracellular milieu of viable tumor cells(Multhoff et. al. (2001) Cell Stress & Chaperones 6, 337). Concomitantwith an increased cytolytic activity, following contact either withHsp70-protein or with Hsp70-peptide TKD the cell surface expression ofthe activating form of the C-type lectin receptor CD94 was enhanced inNK cells. Blocking assays using an inhibitory antibody specific for CD94revealed an involvement of CD94 in the interaction of NK cells withHsp70 membrane-positive tumor cells (Multhoff et. al. (1999) Exp.Hematology 27, 1627). These data indicate that apart from HLA-Epresenting leader peptides of classical HLA-alleles (Lanier et. al.(1998) Immunity 8, 693; Braud et. al. (1998) Nature 391, 795), theC-terminal localized Hsp70-peptide sequence TKD might be considered as apotential ligand for a yet undefined activating CD94 receptor complex.Although the preceding observations indicate that Hsp70-peptidefunctions as a tumor-selective target recognition structure for CD94positive NK cells (Multhoff et al. (1997) J. Immunol. 158, 4341), themechanism by which NK cells lyse Hsp70 positive tumor target cellsremained to be elucidated. In addition, it is desirable to specificallytrigger the lytic activity of NK cells towards tumor cells in a morespecific manner than has hitherto been possible. All these scientificgoals serve as a means to derive more efficacious and more specificapproaches to disease treatment and in particular to tumor treatment.

Surface expression of heat-shock proteins including Hsp70 has beenreported to occur also after viral infection or in response to stress.In particular, membrane Hsp70 was found in HIV-infected lymphoid cells(Di Cesare et al. (1992), Immunology 76, 341) and in HTLV I-infectedrabbit cell lines (Chouchane et al. (1994), J. Infect. Dis. 169, 253).Similarly, it is conceivable that cells infected by bacteria or affectedby inflammation express Hsp70 on their cell surface. Consequently, thelytic activity of NK cells or granzyme B can be directed towards cellsinfected by viruses or bacteria as well as towards cells affected byinflammation.

Thus, the technical problem underlying the present invention was toprovide means and methods for a specific treatment of diseases and inparticular of tumors, viral and bacterial infections and inflammatorydiseases.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly,.the present invention relates to a method of inducing orenhancing the expression of granzyme B in natural killer (NK) cellscomprising contacting NK cells with

-   -   (a) Hsp70 protein;    -   (b) a (C-terminal) fragment of (a) comprising the amino acid        sequence TKDNNLLGRFELSG;    -   (c) a (poly)peptide comprising the amino acid sequence        TKDNNLLGRFELSG; or    -   (d) a combination of (a), (b) and/or (c).

Granzyme B is a serine protease well known in the art and is describedto be involved in the process of apoptosis/programmed cell death (Berke(1995) Cell, 81(1), 9-12; Froelich et. al. (1998) Immunology Today,19(1), 30-26); Metkar S et al. Cytotoxic cell granule-mediatedapopstosis: perforin delivers granzyme B serglycin complexes into targetcells without plasma membrane pore information, Immunity 16 (2002),417-428

This enzyme promotes DNA fragmentation by cleavage of procaspases intotheir activated form and thereby induces programmed cell death through aBcl-2 inhibitable pathway. Granzyme B starts to induce the process ofapoptosis upon presence in the cytosol of a target cell.

The term “NK cells” (“natural killer cells”) comprises large, granularlymphocytes expressing CD45 on the surface and exhibiting killeractivity without prior stimulation. They are particularly characterisedin that they do not express CD3 or T cell receptor α/β- or γ/δ and canbe stimulated by interleukin-2.

The NK cells stimulated by the method of the invention are furthercharacterised by the following properties:

-   -   they are transient plastic-adherent after addition of IL-2 in        amounts of 10 to 10,000 Units, e.g. of 100 I U, wherein IL-2 can        be purchased from the firm Chiron;    -   the adherence takes effect 3-18 hours after addition of IL-2 on        newly isolated PBL (peripheral blood lymphocytes depleted-by        monocytes);    -   the NK cells exhibit a CD16dim expression (average value of        fluorescence weak);    -   the NK cells express CD56 and CD57 as typical NK marker;    -   the NK cells express CD94 (C-type lectin killer cell receptor)    -   the NK cells secrete after activation with Hsp70 and cytokines        IFN gamma;    -   the NK cells can be stimulated by addition of Hsp70, Hsp70        fragment or Hsp70-peptide (purified protein) (growth and        cytotoxic activity);    -   they are not dependent on the patient's MHC type.

According to the invention, other NK-cell populations can be used, too.Further methods for obtaining said population are known in the art andinclude isolation using magnetic beads and cell-sorting. In this case,however, it is a pre-requisite that they can be activated by Hsp70 or bythe above-mentioned fragments or (poly)peptides. According to theinvention, isolated NK cells can be used. It is furthermore possible touse cell mixtures such as peripheral mononuclear blood cells (PBMC)containing NK cells.

In a particularly preferred embodiment of the method of the inventionperipheral blood mononuclear cells (PBMC) or a fraction thereof whichcontain NK cells are L used as physiological cell suspensions.

Using appropriate methods, the NK cells can be obtained from thepatients to be treated or from a healthy donor by taking blood.Preferably, buffy-coats or lymphocyte concentrates obtained by othermeans containing NK cells are to be used.

Buffy-coats or lymphocyte concentrates are taken from patients via theveins and e.g. heparin is added to prevent clotting of the cells. Thebuffy-coats to which heparin has been added are collected in a sterilereceptacle (usually sterile plastic bags) and then centrifuged usingFicoll density centrifugation resulting in an accumulation of bloodcells (=PBMC, peripheral blood mononuclear cells, e.g. lymphocytes,monocytes, granulocytes, and so on). The lymphocyte concentrate remainssterile in sterile culture bags.

The buffy-coats containing peripheral blood mononuclear cells are usedin the form of a physiological cell suspension, preferably with heparinadded. The heparin prevents aggregation of the cells.

Methods for the stimulation of NK cells by incubation with Hsp70proteins of C-terminal fragments thereof have been described in WO 99 49881. Surprisingly it has been found, that expression of granzyme B isinduced or enhanced in NK cells by contacting said cells with Hsp70protein, a fragment thereof comprising the amino acid sequenceTKDNNLLGRFELSG, a (poly)peptide comprising the amino acid sequenceTKDNNLLGRFELSG, or a combination of said proteins/(poly)peptidespreferably in combination with IL-2. Preferably, the fragment referredto above and in connection with other (preferred) embodiments of theinvention is a carboxy-terminal (C-terminal) fragment of Hsp70.

According to the invention, the term “Hsp70 protein” relates toeukaryotic heat-shock proteins (HSPs). The expression of said HSPs canbe induced by heat but also by a number of other reagents such as e.g.amino acid analogues, heavy metals, ionophores or cytotoxines, whereinthe factor of the increase in the expression by means of induction is atleast 5, compared to the constitutive expression. The complete aminoacid sequence has been published in Milner et al. (1990) Immunogenetics32 (4), 242-251.

According to the invention, the term “fragment” of the Hsp70 proteinalso comprises (poly)peptides exhibiting an amino acid sequence from therange of amino acids 384-641 of the human Hsp70. All C-terminal(carboxy-terminal) fragments at least comprise the amino acid sequenceTKDNNLLGRFELSG. Methods for the isolation of corresponding(poly)peptides are known in the art and particularly described in theappended example 1. Thus, the person skilled in the art is also able toproduce fragments from the above-mentioned fragment 384-641 byrecombinant techniques without further ado (standard methods for thisare described in Sambrook et al., “Molecular Cloning, A LaboratoryManual”, 2. edition 1989, CSH Press, Cold Spring Harbor, N.Y.) and testthem for the activation properties wanted.

The term (poly)peptide refers to peptides as well as polypeptides(proteins). According to the conventional understanding, peptidescomprise up to 30 amino acids whereas polypeptides consists of more than30 amino acids. This convention is also employed in accordance with theinvention. Further, in accordance with the invention, the amino acidsthroughout the description are referred to by the one letter code.

In one alternative (poly)peptides comprising the amino acid sequenceTKDNNLLGRFELSG are (poly)peptides consisting of the recited amino acidsequence and optionally further amino acid stretches N-terminally andC-terminally thereof derived from Hsp70, fused to further randomlychosen or naturally occuring amino acid sequences. Thus, the method ofthe present invention relates to the stimulation of NK cells by fusionproteins comprising the sequence of the 14-mer Hsp70-peptide.

A preferred embodiment of the invention relates to a method wherein theHsp70 protein, the (C-terminal) fragment thereof, the (poly)peptidecomprising the amino acid sequence TKDNNLLGRFELSG, or the combinationthereof is in an uncomplexed state.

HSPs are known in the art to occur in complexes with a large number ofdifferent substrates peptides (Tamura et. al. (1997) Science, 278,117-223). However, it has been suprisingly found that heat-shockproteins, (C-terminal) fragments thereof or derivatives derivedtherefrom (see above) induce immunological activities by means ofactivation of NK cells even if they do not form complexes with peptides.

Thus, according to the methods described in WO 99 49 881 the personskilled in the art is able stimulate NK cells using Hsp70 protein or(poly)peptide comprising the amino acid sequence TKDNNLLGRFELSG in anuncomplexed state.

According to a preferred embodiment the method of the invention is an invivo method.

A procedure envisaged would include to inject Hsp70, Hsp70 fragment orHsp70 peptide into patents for in vivo stimulation of NK cells toproduce granzyme B.

According to alternatively preferred embodiments said method is an exvivo method or an in vitro method.

This method comprises isolation of NK cells or a population of cellscomprising NK cells as described herein above, wherein a physiologicalcell suspension containing NK cells is mixed with Hsp70 protein, theC-terminal fragment thereof or a derivative thereof or aprotein/(poly)peptide comprising the amino acid sequence TKDNNLLGRFELSGand incubated to induce or enhance expression of granzyme B in the NKcells.

The incubation can e.g. take place in an incubator, at physiologicaltemperature (37° C.) on a shaker (gentle shaking), at 5% CO₂+>80%humified atmosphere also otherwise retaining physiological conditionsthat allow the survival of NK cells.

A further preferred embodiment of the invention relates to a methodfurther comprising reinfusion of preferably autologous and/or allogeneicNK cells with induced or enhanced granzyme B expression into a mammal.

Once the NK cells have undergone an in vitro or ex vivo treatment toinduce or enhance granzyme B expression, they are re-infused into apatient. Re-infusion can take effect using standard medical equipment.For example, reinfusion of NK cells or PBMC containing NK cells can bei.v., i.p., s.c., or intratumoral.

According to a further preferred embodiment of the invention said mammalis a human.

In another preferred embodiment of the method of the invention saidcontacting of the NK cells with Hsp70 protein, the (C-terminal) fragmentthereof or a derivative thereof or a protein/(poly)peptide comprisingthe amino acid sequence TKDNNLLGRFELSG is effected for at least 12hours. According to a further preferred embodiment said contacting iseffected for at least 4 days.

The present invention relates in another preferred embodiment to amethod wherein said NK cells, prior to said contacting, are obtainedfrom bone marrow cells by incubating said bone marrow cells withinterleukin-15 (IL-15) and stem cell factor (SCF) at concentrations of 1ng/ml-1000 ng/ml per cytokine for at least 7 days up to 4 months.

This preferred embodiment of the invention allows for the freshisolation of NK cells after stimulation of bone marrow cells with thenamed cytokines. The NK cells such obtained display the typical NK cellmarkers referred to herein above. Preferred concentration of cytokinesare in the range of 100 ng/ml for each cytokine. After stimulation withthe cytokines and differentiation into NK cells which display CD94 andCD56 on their surface, contacting of these cells may proceed with Hsp70protein or the above mentioned fragment of (poly)peptide or thecombination of the above as mentioned herein before.

An alternative embodiment of the invention relates to the use of NKcells which produce (i.e. express) granzyme B after stimulation with

-   -   (a) Hsp70 protein;    -   (b) a (C-terminal) fragment of (a) comprising the amino acid        sequence TKDNNLLGRFELSG;    -   (c) a (poly)peptide comprising the amino acid sequence        TKDNNLLGRFELSG; or    -   (d) a combination of (a), (b) and/or (c);        for the preparation of a pharmaceutical composition for the        treatment of tumors, viral and bacterial infections and        inflammatory diseases.

According to the invention, pharmaceutical preparations are defined assubstances and preparations of substances which, when used on or in thehuman body, are meant for healing, alleviating, preventing orrecognising diseases, ailments, physical defects or pathologicaldiscomforts.

Optionally, said pharmaceutical compositions further comprise apharmaceutically acceptable carrier, diluent or adjuvant.

Examples of suitable pharmaceutically acceptable (tolerable) carriersare known to the person skilled in the art and comprise, for example,phosphate-buffered saline solutions, water, emulsions, such as oil/wateremulsions, sterile solutions, and so on. The pharmaceutical compositions(pharmaceutical preparations) containing such carriers may be preparedaccording to common methods. The pharmaceutical compositions may beadministered to the respective individuals in an appropriate dosage.Ways of administration are, for example, intravenous (i.v.),intraperitoneal (i.p.), intratumoral, subcutaneous (s.c.), intramuscular(i.m.), topic or intradermal. The dosage depends on many factors, e.g.on the patient's size, sex, weight, age as well as the type of thecomposition specially administered, the kind of administration and soon. The compositions may be administered locally or systemically.Generally, administration is carried out parenterally. Therefore, the NKcells treated with Hsp70 protein, the C-terminal fragment thereof or aderivative thereof or a protein/(poly)peptide comprising the amino acidsequence TKDNNLLGRFELSG according to the invention are preferablyinjected intravenously. An injection may also be carried out directlyinto the tumour with an effective amount of NK cells being injected.Other known types of application are, of course, also possible. Anoperable number of NK cells administered includes the range of 5×10⁷ to2×10⁹ NK cells, for example, as components of a leukapheresate. In sucha leukapheresate, NK cells are usually present in an amount of between5% and 20%.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous, carriers include water, aqueous solutions,emulsions or suspensions, including saline such as 0.9% NaCl, phosphatebuffered, X-vivo 20 etc. and buffered media. Parenteral vehicles includesodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, or lactated Ringer's. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like. Furthermore, thepharmaceutical composition of the invention may comprise further agentssuch as interleukins or interferons depending on the intended use of thepharmaceutical composition.

According to a preferred embodiment of the invention said NK cells whichare used for the preparation of a pharmaceutical composition for thetreatment of tumors, viral or bacterial infections or inflammatorydiseases are stimulated by the method according to the invention.

Further, according to the invention tumors treated with saidpharmaceutical composition comprise in accordance with the inventiontumor cells which express Hsp70 on the surface of their membrane. Also,cells affected by said infection or inflammation comprise in accordancewith the invention cells which express Hsp70 on the surface of theirmembrane.

Methods for the detection of surface expression of Hsp70 are known inthe art and comprise, e.g. histological methods, flow cytometry etc.

More preferred said tumors are selected from a group consisting ofstomach, gastric, colorectal, pancreas, mammary, lung, gynecological,head and neck cancer, dermatological (e.g. melanoma), neuronal tumors,leukemia and lymphoma.

Preferred viral infections in accordance with the invention areinfections by HIV and Hepatitis virus.

The invention also relates to the use of granzyme B for the preparationof a pharmaceutical composition for the perforin-independent treatmentof tumors, viral or bacterial infections or inflammatory diseases.

A most important aspect of the present invention is mirrored by theabove recited embodiment. In contrast to the speculation of the priorart, it could be shown in accordance with the present invention, thatgranzyme B is effective in the treatment of tumors independent of theperforin-pathway. This has important applications in the strategy oftreating tumors since uptake of the pharmaceutically active compound maynow be deviced independent of the perforin-pathway. Granzyme B could beadministered intratumoral, iv, subcutan.

In a preferred embodiment of the use of the present invention, granzymeB is used as the only pharmaceutically active component in saidpharmaceutical composition.

Again, this preferred embodiment of the invention has importantimplications in the design of the necessary components of thepharmaceutical composition to be used in the treatment of cancers.Importantly, there is no need to include further pharmaceutically activeingredients into the pharmaceutical composition in order to effectivelytreat tumors and/or reduce the size of the tumors or to treat viral orbacterial infections or inflammatory diseases.

In addition, the present invention relates to a method of treatingtumor, viral or bacterial infections or inflammatory diseases comprisingsteps of:

-   -   (a) contacting NK-cells with tumor cells bearing Hsp70 on their        surface or cells affected by said infection or inflammation amd        bearing Hsp70 on their surface;    -   (b) allowing granzyme B to enter said cells via ion channels        formed by said Hsp70 on the tumor cell surface; and    -   (c) allowing said cells to undergo apoptosis as a result of the        enzymatic activity of granzyme B.

As an alternative, granzyme by may be directly applied instead of beingexpressed by stimulated NK cells. Accordingly, the present inventionrelates to a method of treating tumor, viral or bacterial infections orinflammatory diseases comprising steps of:

-   -   (a) contacting tumor cells bearing Hsp70 on their surface or        cells affected by said infection or inflammation and bearing        Hsp70 on their surface with granzyme B;    -   (b) allowing granzyme B to enter said cells via ion channels        formed by said Hsp70 on the cell surface; and    -   (c) allowing said cells to undergo apoptosis as a result of the        enzymatic activity of granzyme B.

Ranges of tumor cells or cells affected by said infection orinflammation and concentrations of granzyme B as well as time ranges forthe contact of granzyme B and tumor cells or cells affected by saidinfection or inflammation can be derived from the person skilled in theart by studying the above recited teachings of the invention.

In a preferred embodiment of this method of the invention, granzyme B isadministered in a final concentration of 1 μg/ml to 500 μg/ml.

In another preferred embodiment of this method of the invention,granzyme B is administered in a final concentration of 1 ng/ml to 10ng/ml. It is most preferred that granzyme B is administered in a finalconcentration of about 6 ng/ml. In this regard it is important to notethat modes of administration are most preferred that deliver theseconcentrations of granzyme B directly to the tumor cells or cellsaffected by said infection or inflammation.

In another preferred embodiment of the use of the present invention orthe method of the present invention, granzyme B is delivered/packaged ina liposome. Encapsulation of pharmaceutically active compounds inliposomes is well established in the art.

The figures show:

FIG. 1 shows the identification of granzyme B as the interacting partnerfor Hsp70-protein and Hsp70-peptide TKD.

FIG. 1A: Hsp70-protein (Hsp70), bovine serum albumin (BSA) andHsp70-peptide (TKD) columns were incubated with either cell lysates ofthe NK-cell line YT or the non-NK cell line K562. Bound proteins wereeluted from the columns in 5 fractions (F1-F5), resolved on a SDS-PAGE.Following silver stain, eluates of YT cells derived from Hsp70 and TKDcolumns, revealed a dominant 32 kDa protein band in fractions two (F2)and three (F3). No 32 kDa protein band was detectable in YT eluatesderived from BSA columns and in K562 eluates derived from TKD columns.The position of the 32 kDa band is indicated with an arrowhead.

FIG. 1B: The tryptic peptides of the Coomassie-blue stained 32 kDa bandof fraction 3 (F3), derived from the TKD column, correspond to humangranzyme B. The probability of identification was 100% and the estimatedZ-score was 1.89 corresponding to >95% confidence.

FIG. 1C: Corresponding Western blot analysis of YT and K562 cell eluates(F3) following incubation with Hsp70-protein (Hsp70) and Hsp70-peptide(TKD) columns. The blot was autoradiographed and the localization ofgranzyme B was visualized by immunostaining with the granzyme B specificmAb 2C5. Eluates of YT cells (left), but not of K562 cells (right)revealed a 32 kDa granzyme B protein band.

FIG. 1D: Intracellular flow cytometry of permeabilized YT cells (left)and K562 cells (right) using the phycoerythrin (PE)-conjugated granzymeB specific monoclonal antibody HC2-PE (solid line), as compared to anisotype-matched negative control antibody (dashed line). Only YT cells,but not K562 cells, contain cytoplasmic granzyme B.

FIG. 2 shows specific cell surface binding and uptake of granzyme B(grB) by Hsp70 membrane-positive tumor cells

FIG. 2A: Comparative binding of granzyme B (2 μg/ml) to the cell surfaceof CX+/Cx− and Colo+/Colo− tumor cells at 4° C., and uptake into thecytosol after a temperature shift to 37° C. for 30 min, using thephycoerythrin (PE)-conjugated granzyme B specific monoclonal antibodyHC2-PE. First row, light microscopy, second row, immunofluorescence ofcells without granzyme B (control); third row, immunofluorescence ofcells after addition of granzyme B, as specified (grB). Onerepresentative fluorescence microscopy of three showing identicalresults is illustrated; magnification 40×.

FIG. 2B: Intracellular flow cytometry of permeabilized CX+/CX− (n=2) andColo+/Colo− (n=4) tumor cells using granzyme B specific monoclonalantibody HC2-PE before (dashed line) and after (solid line) incubationof the tumor cells with 1 μg/ml, 2 μg/ml, 4 μg/ml granzyme B at 37° C.for 30 min. Only CX+ and Colo+, but not CX− and Colo− cells showed adose-dependent shift of the granzyme B peak to the right, indicatinguptake of extracellular offered granzyme B.

FIG. 3 shows an experiment in which apoptosis is selectively induced byisolated granzyme B (grB) in Hsp70 membrane-positive tumor cells

FIG. 3A: Percentage of Annexin V-FITC positive and propidium iodide (PI)negative CX+/Colo+ (left) and CX−/Colo− (right) cells, either untreated(black bars), or following incubation either with camptothecin (4 μg/ml;light grey bars) or granzyme B (6 ng/ml; dark grey bars) for 4 h, 12 h,and 24 h. The data represent the mean of three to four independentexperiments±standard deviation; * marks values significantly differentfrom control (p<0.05).

FIG. 3B: A representative flow cytometric analysis of Annexin V-FITCpositively and propidium iodide (PI) negatively stained CX+ and CX−cells, either untreated (control), or following incubation with granzymeB (grB) for 24 h. The percentage of Annexin V-FITC positively stainedcells is given in percentage in the lower right corner of each graph.

FIG. 3C: Light microscopical analysis of adherent growing CX+/CX− andColo+/Colo− tumor cell clusters either untreated (control) or followingtreatment with camptothecin (cam, 4 μg/ml) or granzyme B (grB, 10ng/ml), for 24 h. Scale bar indicates 100 μm.

FIG. 3D: In parallel, either untreated (control), camptothecin (cam) orgranzyme B (grB) treated CX+/CX− and Colo+/Colo− cells (24 h) werestained with DAPI. Considerable nuclear DNA fragmentation was observedin all tumor sublines following incubation with camptothecin (middlepanel). After incubation with granzyme B only CX+ and Colo+ cellsexhibited nuclear DNA fragmentation (lower panel, left). No signs ofapoptosis was observed in CX− and Colo− cells following incubation withgranzyme B (lower panel, right). Scale bar represents 10 μm.

FIG. 4 shows an experiment in which kill of Hsp70 membrane-positivetumor cells is demonstrated. Apoptosis mediated by granzyme B positiveNK cells is blockable by Hsp70 specific mAb.

FIG. 4A: Light microscopy (magnification 20×) of Hsp70 membrane-positiveCX+ and Hsp70 membrane-negative CX− cell colonies, either untreated(control) or following a 12 h co-incubation with Hsp70-peptide TKDstimulated NK cells (+NK). The effector to target cell ratio (E:T) was20:1. Scale bar represents 200 μm, the insert in the lower right cornerof each graph shows one representative cell colony, magnification 2.5×.

FIG. 4B: Cell kill of CX+/Colo+ (left panel) and CX−/Colo− (right panel)tumor target cells by naive (NK d0) and Hsp70-peptide TKD stimulated NKcells (NK d3) was quantified in ⁵¹Cr release assays. Intracellulargranzyme B levels in naive NK cells (NK d0) versus TKD stimulated NKcells (NK d3) was 1.4-fold increased; concomitantly, lysis of CX+ cellswas elevated 1.5-fold, that of Colo+ cells 2.0-fold at differenteffector to target ratios. Lysis of CX− and Colo− tumor cells remainedunaffected.

Furthermore, the increased cytolytic activity of TKD stimulated NK cells(NK d3 Hsp70 mAb) against CX+ and Colo+ cells was completely inhibitedby Hsp70 specific antibody down to the level of lysis of Hsp70membrane-negative tumor cells (1.7-fold inhibition, dotted line).Cytotoxicity was determined at E:T ratios ranging from 2:1 to 20:1;spontaneous release of each target cell was below 10%. The datarepresent the mean of three independent experiments±standard deviation.

EXAMPLES

The following examples illustrate the invention. These examples shouldnot be construed as limiting: the examples are included for purposes ofillustration and the present invention is limited only by the claims.

Example 1 Materials and Methods

Cells

The NK cell line YT was cultured at cell densities ranging from0.1-0.5×10⁶ cells/ml RPMI-1640 medium (Life Technologies, Eggenstein,Germany) containing 10% heat inactivated fetal calf serum (FCS, LifeTechnologies, Eggenstein, Germany) supplemented with 6 mM L-glutamine,and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin; LifeTechnologies). Transient plastic adherent NK cells were derived frombuffy coats of healthy human volunteers. Ficoll separated peripheralblood mononuclear cells (PBMC) were cultured in rIL-2 (100 IU/ml,Chiron, Frankfurt, Germany) for 12 h. Following adherence selection ofmonocyte depleted peripheral blood lymphocytes according to a modifiedmethod of Vujanovic (Vujanovic et. al. (1993) Cell. Immunol. 151, 133),cells were cultivated in RPMI-1640 medium supplemented withHsp70-peptide TKD (2 μg/ml) for 3 days.

The human tumor sublines CX+/CX− and Colo+/Colo− were derived by cellsorting of CX-2 colon (Hsp70 positive: 60%) and Colo357 pancreas (Hsp70positive: 70%) carcinoma cell lines using the Hsp70 specific monoclonalantibody C92F3B1, according to a previously described protocol (Multhoffet. al. (1997) J. Immunol. 158, 4341). Consequently, CX+ and CX− areautologous, as are Colo+ and Colo− Hsp70 stably high expressing CX+(Hsp70 positive: 80%) and Colo+ (Hsp70 positive: 85%) carcinoma sublinesdiffer significantly from Hsp70 stably low expressing CX− (Hsp70positive: 25%) and Colo− (Hsp70 positive: 35%) carcinoma cells.

The carcinoma sublines CX+/Colo+ and CX−/Colo−, that differ with respectto their membrane expression of Hsp70, but exhibit identical MHC class Iexpression, and the leukemic non-NK cell line K562 were cultured inRPMI-1640 medium supplemented with 5% FCS, 6 mM L-glutamine andantibiotics. Exponentially growing tumor cells (day 1 after cellpassage) were used for granzyme B, camptothecin treatment and as targetsin cytotoxicity assays.

All cell lines were screened regularly for mycoplasma contaminations byan enzyme-immunoassay detecting M. arginini, M. hyorhinis A. laidlawii,and M. orale (Roche, Mannheim, Germany). Only mycoplasma-free cell lineswere used.

Affinity Chromatography and Immunoprecipitation

Bovine serum albumine (BSA, 1 mg/ml, Sigma-Aldrich, Steinheim, Germany),1 mg/ml lyophylized, recombinant human Hsp70-protein (Stressgen, BritishColumbia, Canada) or 2 mg/ml Hsp70-peptide TKD (TKDNNLLGRFELSG,aa₄₅₀₋₄₆₃, Bachem, Bubendorf, Switzerland) were incubated withequilibrated AminoLink agarose beads (Pierce, Rockford, USA) in 2 ml for6 h, together with the reductant NaCNBH₃. Binding capacity of BSA,Hsp70-protein and Hsp70-peptide TKD was greater 95%. Following removalof uncoupled material by extensive washing with Tris-buffer andquenching of non-reactive groups, cell lysates were administered to theBSA, Hsp70-protein and Hsp70-peptide TKD conjugated columns for 1 h.

After washing with 10 column volumes of 20 mM Tris buffer, boundproteins were eluted with 3 M sodium chloride in 20 mM Tris buffer, in 5fractions. Each fraction was subjected to a 10% SDS-PAGE and blotted toPVDF membranes.

Membrane Preparation

Membrane purification was performed by dounce homogenization of 50×10⁶cells in hypotonic, EDTA-free buffer containing the protease inhibitorPMSF followed by sequential centrifugation at 1,000 g for 5 min and at100,000 g, at 4° C., for 60 min. The pellet containing membranes wasresuspended in 2 ml 0.3 M NaCl in 50 mM Tris buffer, 0.5% NP40, pH 7.6.

Western Blot Analysis

Following blocking in skim-milk (0.1%) and incubation with mAb directedagainst granzyme B 2C5 (IgG2a, Becton Dickinson, Heidelberg, Germany),at 4° C., for 5 h, Western blots were washed and incubated with asecondary mouse anti-IgG HRP Ab (Dianova, Hamburg, Germany), for 1 h.Proteins were detected using the ECL kit (Amersham Bioscience) for 5sec.

Protein Identification by Peptide Mass Fingerprinting

The Hsp70-protein and Hsp70-peptide TKD precipitated 32 kDa protein bandwas cut out from Coomassie-blue stained gels, digested with trypsin, anddesalted using reversed phase ZIP tips (Millipore, Eschborn, Germany.The samples were embedded in 4-hydroxy-α-cyano-cinnamic acid and thepeptide masses were determined with a Perseptive Voyager DePro MALDI-TOF(Matrix Assisted Laser Desorption Ionisation—Time Of Flight) massspectrometer in reflective mode. A peaklist was compiled with the m/zsoftware (Proteometrics) and used for peak selection; the resultingpeptide mass fingerprint was used to search the non-redundant NCBIprotein database using the Profound search engine (Proteometrics).Granzyme B was identified with 100% probability and >95% confidence.

Flow Cytometry

Cells (0.5×10⁶) were fixed in paraformaldehyde (1% PFA in PBS) for 10min, and permeabilized in PBS containing BSA (0.5%), NaN₃ (0.1%), andsaponin (0.1%). Then permeabilized cells were incubated either with thegranzyme B-phycoerythrin conjugated monoclonal antibody HC2-PE (IgG1;Hölzel Diagnosbka, Cologne, Germany) with an isotype-matched IgG1control antibody, at 4° C. for 1 h, in the dark. Following washing cellswere analysed on a FACSCalibur instrument (Becton Dickinson, Heidelberg,Germany).

Treatment

Stock solutions of camptothecin (4 mg/ml, Sigma, Munich, Germany) werediluted in DMSO and stored at 4° C. in the dark. Granzyme B (6 ng/ml,Hölzel Diagnostics, Cologne, Germany) solutions were freshly prepareddirectly before usage. Exponentially growing cells (0.5-1.5×10⁶/ml) wereincubated either with camptothecin at a final concentration of 4 μg/mlor with purified, enzymatically active granzyme B (10 ng/ml, 1 μg/ml, 2μg/ml; 4 μg/ml) (Shi et. al. (2000) Methods in Enzymology 322, 125) for10 min, and 30 min either at 4° C. or at 37° C. After washing inRPMI-1640 medium binding and uptake was determined in nonpermeabilizedand permeabilized tumor cells by flow cytometry and fluorescencemicroscopy on a Axioscop 25 scanning microscope (Zeiss, Jena, Germany)equipped with a 40× objective and standard filters. Images were treatedby multiplicative shading correction using the software Axiovison (ZeissVison, Jena, Germany). Granzyme B was visualized in red by using theHC2-PE antibody.

Apoptotic Cell Death was Determined after Incubation of Tumor Cells with6 ng/ml Granzyme B for 4 h, 12 h, and 24 h at 37° C. and Washing inRPMI-1640 Medium by Different Apoptosis Assays, as Described Below.Apoptosis Assays

Annexin V-FITC staining: Briefly, cells were washed twice in Hepesbuffer containing 5 mM CaCl₂ and incubated with Annexin V-FITC (Roche)for 10 min at room temperature. Annexin V-FITC positively stained cellswere measured on a FACSCalibur flow cytometer (Becton Dickinson,Heidelberg, Germany).

DAPI-staining: Methanol/aceton fixed cells (0.1×10⁶ cells/100 μl) wereincubated with 0.5 μg/μl 4,6-diamino-2-phenylindole (DAPI) inPBS/glycerol (3:1), for 15 min in the dark. Following washing in PBS thecells were mounted with Fluorescent Mounting Medium (Dako, Glostrup,Denmark) and than analysed for fluorescence using a Zeiss model Axioscop2 scanning microscope (Zeiss Jena, Germany) equipped with a 40×objective and standard filters. Apoptosis was visualized withDAPI-staining in 50 cells, each. Images were treated by multiplicativeshading correction using software Axiovision (Zeiss Vision Jena,Germany).

Cytochrome c release: Cytochrome c release was determined using aquantitative immunoassay; (DCDCO, R&D Systems, Wiesbaden, Germany).Briefly, either untreated, camptothecin (4 μg/ml) or granzyme B (6ng/ml) treated CX+ and CX− cells (1.5×10⁶/ml) were washed in PBS andtreated with lysis buffer for 1 h at room temperature. Followingcentrifugation at 1,000 g for 15 min supernatants were removed and 200μl of a 1:100, 1:250, and a 1:500 dilution was used for a sandwichELISA. Following incubation with substrate solution in the dark for 30min the reaction was stopped. The optical density of each well wasdetermined on an ELISA reader at 450 nm. The amount of cytochrome c wasdetermined according to a calibration curve.

Granzyme B ELISPOT

Granzyme B release was compared in unstimualted NK cells (NK d0) and TKDstimulated NK cells (NK d3) after a 4 h co-incubation period with tumorcell lines CX+/CX− and Colo+/Colo−, at different effector to target cellratios (E:T) ranging from 20:1 to 2:1. For detection a Granzyme BELISPOT kit (#552572, BD, Heidelberg, Germany) was used. Briefly,96-well ELISPOT plates (MAIPN45, Millipore) were coated overnight at 4°C. with capture antibody, blocked with RPMI-1640 culture mediumcontaining 10% FCS and incubated with tumor and effector cells for 4 hat 37° C., as specified before. After washing in deionized water andwash buffers A and B, biotinylated anti-granzyme B antibody was added (2μg/ml) for 2 h. After another two washing steps granzyme B wasvisualized by the addition of freshly prepared avidin-horseradishperoxidase (2 h) and substrate solution (25 min incubation period).Spots were counted automatically using ImmunoSpot Series I Analyzer.⁵¹Cr Release Assay and Inhibition Assay

NK cell mediated cytotoxicity was measured using a 12 h ⁵¹Crradioisotope assay. As target cells the colon carcinoma sublines CX+ andCX− were used. For blocking studies the mAb C92F3B1 and an isotypematched control antibody (IgG1) were used at a final concentration of 5μg/1×10⁶ cells. Following incubation of CX+ and CX− target cells withthe antibodies for 30 min at 4° C., the cells were labeled with ⁵¹Cr andthe cytotoxicity assay was performed as described by MacDonald(MacDonald et. al. (1974) J. Exp. Med. 140,718). The percentage ofspecific lysis was calculated as: [(experimental release−spontaneousrelease)/(maximal release−spontaneous release)]×100.

Example 2 Granzyme B is an Interacting Partner of Full Length Heat ShockProtein 70 (Hsp70) and of Hsp70-peptide TKD

Partner proteins were identified by affinity chromatography onimmobilized human Hsp70-protein (1 mg) or a 14 amino acid peptide,coined TKD (TKDNNLLGRFELSG, aa₄₅₀₋₄₆₃, 2 mg), containing theextracellular epitope of Hsp70 that was found mediating the interactionwith NK cells. This peptide was previously identified as the epitope(Reineke et. al. (1-996) Immunobiol. 196, 96) of an Hsp70 specificantibody (Welch and Suhan (1986) J. Cell. Biol. 103, 2035), whichspecifically detects membrane-bound Hsp70 on viable tumor cells(Multhoff et. al. (1995) Int. J. Cancer 61,272; Multhoff et. al. (1995)Blood 86,1374). A cell lysate of the NK cell line YT (Drexler et. al.(2000) Leukemia 14,777) was fractionated on immobilized Hsp70 or TKDpeptide columns. The material bound to the columns was eluted with 3 Msodium chloride within five fractions. As controls, YT cell lysates wereadministered to carrier or BSA-conjugated columns. Moreover, lysate of anon-NK cell line (K562) was fractionated on TKD-conjugated affinitycolumn. The eluted fractions were separated by SDS-PAGE (10%) andvisualized by silver-staining. A dominant protein band of apparentmolecular weight of 32 kDa was observed in fractions two (F2) and three(F3) of YT cell eluates derived from the Hsp70-protein (Hsp70) and theHsp70-peptide (TKD) column (FIG. 1A, YT). This band was not observed ineluates of unconjugated sepharose columns (data not shown) orBSA-conjugated colums nor in the material eluted from the TKD affinitycolumns loaded with K562 cell lysates (FIG. 1A,). Identical results wereobtained with Hsp70-protein columns (data not shown). In parallel, theeluates of Hsp70-peptide TKD and Hsp70-protein derived from fraction 3(F3) were separated by SDS-PAGE and stained with Coomassie-blue. The 32kDa protein band derived from F3 of the Hsp70-peptide column was cut outand digested with trypsin (FIG. 1B). The resulting peptides wereanalyzed by MALDI-TOF peptide mass fingerprinting. Sequence of thetryptic peptides exhibited 100% homology with granzyme B with anestimated Z-value was 1.89 indicating a probability of greater 95% forgranzyme B (FIG. 1B). The identity of the 32 kDa protein band asgranzyme B was further confirmed by Western blot analysis using thegranzyme B specific antibody 2C5 (IgG2a): YT cell eluates obtained fromHsp70-protein (Hsp70) and Hsp70-peptide (TKD) columns, both revealed adominant 32 kDa granzyme B protein band (FIG. 1C). Granzyme B was notdetected in eluted fraction of Hsp70 or TKD affinity columns loaded withK562 cell lysates (FIG. 1C). Flow cytometry using a phycoerythrin(PE)-conjugated granzyme B antibody (IgG1) again granzyme B showedpositive staining for cytoplasmic granzyme B in YT cells, but not K562cells (FIG. 1D). These observations corroborate our previous results. Insummary, these data indicate granzyme B is a potential partner proteinof Hsp70. Granzyme B is likely to interact with the C-terminal region ofHsp70 termed TKD.

Example 3 Specific Binding and Internalization of Granzyme B in Hsp70Membrane-Positive Tumor Cells

The preceding findings posed the question whether membrane-bound Hsp70might enable specific binding and entry of granzyme B into the cytosol.Therefore, perforin-free, purified granzyme B was co-incubated withtumor cell sublines CX+/CX− and Colo+/Colo− that exhibit differentialHsp70 membrane expression. A light microscopical analysis of untreatedCX+ and Colo+ cells (control) at 4° C. versus 37° C. is shown in theupper row of each panel (FIG. 2A). The corresponding immunofluorescencemicroscopy of the cells at 4° C. and 37° C. is illustrated below(control). Initially, none of the cells showed any granzyme B staining,neither on the cell surface nor in the cytoplasm. However, after a 15min incubation period of the cells with purified granzyme B (gr B) at 4°C., a ring-shaped fluorescence, indicating a typical cell surfacestaining, was detected on Hsp70 membrane-positive CX+ and Colo+ tumorsublines (FIG. 2A, left panel). A temperature shift from 4° C. to 37° C.during the 30 min incubation period resulted in uptake of granzyme B, asdetermined by a cytoplasmic staining pattern in CX+ and Colo+ tumorsublines (FIG. 2A, right panel). In contrast, the Hsp70membrane-negative counterparts CX− and Colon− either exhibited anygranzyme B cell surface binding at 4° C. nor uptake at 37° C. (data notshown). Flow cytometry analysis of permeabilized cells revealed a faintshift of the granzyme B peak to the right selectively in Hsp70membrane-positive CX+ and Colo+, but not in CX− and Colo− tumorsublines, if the cells were co-incubated with 1 μg/ml granzyme B for 30min at 37° C. (FIG. 2B, upper graph). A dose-dependent increase ingranzyme B uptake, in Hsp70 membrane-positive tumor cells (CX+/Colo+)was detected after co-incubation with 2 μg/ml and 4 μg/ml granzyme B(FIG. 2B, lower graph). However, even at the highest concentration of 4μg/ml, granzyme B was internalized much more pronounced by Hsp70membrane-positive as compared to Hsp70-negative tumor cells (CX−/Colo−).Potential ion channels formed by Hsp70 may play a role in the mechansimof selective granzyme B uptake in Hsp70 membrane-positive tumor cells.Indeed, a particular ion conductance pathway was observed afterincorporation of vesicles derived from purified phospholipids of Hsp70membrane-positive (CX+) tumor sublines. This was not seen in vesiclesobtained from Hsp70 membrane-negative (CX−) tumor cells (data notshown). Based on these results one might speculate about an ion channelactivity facilitating uptake of granzyme B, selectively into Hsp70membrane-positive tumor cells.

Example 4 In vitro Provided Granzyme B Induces Apoptosis Selectively inHsp70 Membrane-Positive Tumor Cells

The preceding findings posed the question whether purified granzyme Bcan induce apoptosis of tumor cells that present Hsp70 on their cellsurface. Hsp70 membrane-positive (CX+/Colo+) and negative (CX−/Colo−)colon carcinoma cells, that exhibit an identical MHC class I expression(Multhoff et. al. (1997) J. Immunol. 158,4341), were incubated for 4 h,12 h, and 24 h with isolated enzymatically active granzyme B (6 ng/ml)(Shi et. al. (2000) Methods in Enzymology 322, 125). As a positivecontrol for apoptosis all cell types were incubated with thetopoisomerase inhibitor camptothecin at a concentration of 4 μg/ml.Apoptosis was determined by Annexin V-FITC staining measured by FACS(FACSCalibur, Becton Dickinson, Heidelberg, Germany), DAPI staining andmitochondrial cytochrome c release. Apoptosis was not detected in CX+and CX− cells incubated with camptothecin (cam) or granzyme B (grB) for4 h incubation (FIG. 3A). CX+ and Colo+ as well as CX− and Colo− cellsincubated with camptothecin underwent apoptosis after a 12 h and 24 hincubation period with camptothecin. It appeared that the coloncarcinoma sublines CX+/CX− are better protected towards acamptothecin-mediated cell death as compared to the pancreas carcinomasublines Colo+/Colo−.

As shown in FIG. 3A, after 12 h the amount of Annexin V-FITC positivelystained CX+ cells increased from 16.6% to 28.0% (1.7-fold) and after 24h from 18% to 39% (2.1-fold). In CX− cells the amount of Annexin V-FITCpositively stained cells increased similarly from 12.1% to 19.8%(1.6-fold) after 12 h, and from 11.9% to 25.1% (2.1-fold) after 24 h. Incontrast, apoptosis was observed selectively in Hsp70 membrane-positiveCX+ cells, incubated with granzyme B for 12 h and 24 h, with an increaseof 16.6% to 20.9% (1.3-fold), and of 18% to 30.2% (1.8-fold),respectively. Similarly, a 1.3-fold and a 2.4-fold increase in theamount of Annexin V-FITC positively stained Colo+ cells were observedafter 12 h and 24 h, respectively. In CX− and Colo− cells neither a 12 hnor a 24 h treatment with granzyme B induces apoptosis. A comparativeAnnexin V-FITC staining pattern of CX+ and CX− cells treated withgranzyme B for 24 h, is illustrated in FIG. 3B. Compared to untreatedcontrol cells (18%), the amount of Annexin V-FITC positively andpropidium iodide (PI) negatively stained CX+ cells increased 1.7-fold(30%) following treatment with granzyme B. However, the amount ofapoptotic CX− cells remained unaltered before and after identicaltreatment with granzyme B. In addition to CX+ cells, granzyme B inducesapoptosis also in the Hsp70 membrane-positive leukemic cell line K562(data not shown). The amount of Annexin V-FITC positively stained K562cells increased from 8.7% up to 16% (1.8-fold).

In order to exclude apoptosis initiated by anoikes light microscopicalanalysis of untreated (control), camptothecin (cam) and granzyme B (grB)treated CX+/CX− and Colo+/Colo− tumor cells were performed. As shown inFIG. 3C, 24 h post-treatment with granzyme B, neither Hsp70membrane-positive nor -negative tumor cell lines exhibited any signs ofloss in plastic adherence. Regarding these findings we ruled out thepossibility that anoikes might be a possible mechanism for the inductionof apoptotic cell death in Hsp70 membrane-positive tumor sublines. It isimportant to note that all apoptosis assays were determined within theadherent cell population following a short term (<1 min) trypsinization.

Consistent with the results from Annexin V-FITC staining pattern allcell types, CX+/CX−, Colo+/Colo− exhibited nuclear fragmentation, atypical sign of apoptosis at a later stage, as detected by DAPI-stainingof nuclear DNA following treatment with camptothecin (4 μg/ml) for 24 h(FIG. 3D, cam). However, DNA fragmentation was detected only in Hsp70membrane-positive CX+ and Colo+ tumor cells after 24 h of granzyme Btreatment (6 ng/ml), whereas no DNA fragmentation was observed in Hsp70membrane-negative CX− and Colo− cells (FIG. 3D, grB).

As an additional test for apoptotic cell death, cytochrome c release wasmeasured following incubation of CX+ and CX− cells with granzyme B for24 h. As summarized in Table I, following incubation with granzyme B (6ng/ml) for 24 h, cytochrome c concentration was elevated from 0.382mg/ml to 0.690 mg/ml (1.8-fold) in CX+ cells. However, no increase incytochrome c was observed in CX− cells following treatment with granzymeB (0.452 mg/ml versus 0.425 mg/ml). In contrast, an incubation withcamptothecin (4 pg/ml) for 24 h, results in a comparable 1.5-foldincrease in cytochrome c concentrations in both cell types. Theseresults indicate that isolated granzyme B induces apoptotic cell deathselectively in tumor cells presenting Hsp70 on their cell surface. Itwas propose that the trigger of apoptosis by granzyme B is mediated viathe extracellular exposed Hsp70 epitope TKD. TABLE I Quantitativedetermination of human cytochrome c in CX+ and CX− tumor cells eitheruntreated (control), or following incubation with camptothecin (4 μg/ml)or granzyme B (6 ng/ml) for 24 h. The data represent the mean of 4independent experiments ± S.E.; * marks values significantly differentfrom control (p < 0.05). cytochrome c (mg/ml) fold increase cellscontrol camptothecin granzyme B CX+ 0.382 ± 0.02 0.555 ± 0.04* 0.690 ±0.08* 1.0 1.5 1.8 CX− 0.452 ± 0.02 0.672 ± 0.02* 0.425 ± 0.075 1.0 1.50.9

Example 5 Stimulation of NK Cells with Hsp70-peptide TKD Induces theProduction of Granzyme B and Increases Kill of Hsp70 Membrane-PositiveTumor Target Cells

The physiological role of our findings was tested in functional assaysusing naive and Hsp70 stimulated human NK cells. Previously, it wasshown that incubation of NK cells with Hsp70-protein at concentrationsbetween 10 and 50 μg/ml or with equivalent Hsp70-peptide concentrations(0.2-2.0 μg/ml) resulted in increased cytolytic activity of NK cellsagainst Hsp70 membrane-positive tumor target cells. Concomitantly, theexpression of the killer cell activating C-type lectin receptor CD94 wasupregulated (Multhoff et. al. (1999) Exp. Hematology 27,1627; Gross et.al. (2002) submitted). Although Hsp70 acts as a tumor-selectiverecognition structure for NK cells, as determined by antibody blockingstudies (Multhoff et. al. (1997) J. Immunol. 158,4341; Multhoff et. al.(1995) Blood 86,1374), the NK-cytotoxic mechanism remains unclear. Toelucidate the possible mechanism, NK cells were incubated withHsp70-peptide TKD (2 pg/ml) for 3 days. A significantly elevatedintracellular granzyme B expression, as determined in 3 independentexperiments, was observed. In contrast, granzyme B expression was notincreased in CD3 positive T cells treated with Hsp70-peptide TKD. Lightmicroscopical analysis of Hsp70-peptide activated NK cells co-incubatedwith Hsp70 membrane-positive CX+ and Hsp70 membrane-negative CX− cellsis illustrated in FIG. 4A. Hsp70 membrane-positive CX+ and Hsp70membrane-negative CX− tumor cells (0.1×10⁶ cells/ml) were cultured induplicates in 24 well plates for 2 days. The proliferation rate of bothtumor cell lines was comparable, as determined by identical cell counts(0.3×10⁶ cells/ml). CX+ and CX− tumor cells in the upper panel werecultured in the absence of NK cells; tumor cells in the lower panel wereco-cultured for 12 h with NK cells that had been stimulated withHsp70-peptide TKD (2 μg/ml, 3 days). Nearly 100% of the CX+ cellcolonies were found in clusters with NK cells and viability of CX+ tumorcells appears to be reduced. In contrast, CX− tumor cells and NK cellswere not found in clusters; Hsp70 membrane-negative CX− tumor cells didnot attract NK cells. Cell viability of CX− tumor cells followingcontact with NK cells appears to be less affected, as compared to thatof CX+ tumor cells. The inserts in the lower right corner of each graphillustrates a 2.5× magnification of one representative cell colony.

Cell kill of CX+/CX− and Colo+/Colo− tumor cells following contact withfreshly isolated, unstimulated (NK d0) or Hsp70-peptide TKD stimulatedNK cells (NK d3) was quantitated in a 12 h ⁵¹Cr release assay (FIG. 4B).Consistently with what was observed in light microscopy (FIG. 4A), thecytolytic activity of TKD stimulated NK cells (NK d3) against CX+ targetcells (left) was significantly enhanced as compared to CX− target cells(right). Concomitant with the increased granzyme B levels followingstimulation with Hsp70-peptide TKD for 3 days, the cytolytic responseagainst Hsp70 membrane-positive CX+ and Colo+ cells, but not againstHsp70 membrane-negative CX− and Colo− cells was significantly elevated;1.5-fold (CX+) and 2.0-fold (Colo+) at E:T ratios of 2:1 to 20:1. SinceCX+ and CX− tumor cells differ only with respect to their Hsp70 membraneexpression but exhibit an identical MHC class I expression pattern theinhibitory effect mediated by killer cell inhibitory receptors (KIR)could be excluded. The increased cytolytic activity against. Hsp70membrane-positive CX+ tumor cells (left), but not against Hsp70membrane-negative CX− tumor cells (right), could be completely inhibitedby pre-incubation of the target cells with the Hsp70 specific monoclonalantibody that is known to detect membrane-bound Hsp70-peptide TKD onviable tumor cells (Multhoff et. al. (1995) Int. J. Cancer 61, 272). Incontrast, the lower lysis of Hsp70 membrane-negative tumor cellsremained unaffected after incubation with Hsp70 antibody. Technically,it is not possible to quantify the absolute amount of granzyme B that istransferred from NK cells into tumor cells by cell-to-cell contact.However, relative values of granzyme B release could be determined byELISPOT analysis. Therefore, a comparison of the cytolytic response offreshly isolated, unstimulated (NK d0) and TKD-stimulated NK cells (NKd3) against CX+/CX− and Colo+/Colo− tumor cells was performedconcomitantly with the definition of the granzyme B release.Irrespectively of the tumor cell line and the E:T cell ratio,co-incubation of tumor cells with unstimulated NK cells (NK d0), alwaysresults in very low granzyme B release; the number of spots was alwaysless than 20. After a 3 day stimulation period with TKD (NK d3) followedby a 4 h coincubation time with tumor cells, granzyme B release wassignificantly upregulated. At an E:T ratio of 5:1, the number ofgranzyme B spots, as determined in three independent experiments, was asfollows: CX+ 260±20, CX− 165±6; Colo+ 137±55; Colo− 66±8. Concomitantly,Hsp70 membrane-positive tumor target cells (CX+/Colo+), were lysedsignificantly better as compared to their -negative counterparts(CX−/Colo−). These data strongly suggest that lysis of Hsp70membrane-positive tumor cells by TKD-activated NK cells is associatedwith granzyme B release. It was hypothesized that interaction ofgranzyme B with membrane-bound Hsp70-peptide TKD is key for its uptakeinto tumor cells and for the induction of apoptosis. When NK cells wereremoved by washing with PBS and the tumor cells were stained with DAPI.Hsp70 membrane-positive CX+ cells showed DNA fragmentation, whereasHsp70 membrane-negative CX− cells not following co-incubation with NKcells. Identical results were obtained with Annexin V-FITC staining(data not shown). These observations strongly suggests that TKDactivated NK cells kill Hsp70 membrane-positive CX+ cells by inductionof apoptosis, which also have elevated levels of granzyme B.

1. A method of inducing or enhancing the expression of granzyme B innatural killer (NK) cells comprising contacting NK cells with (a) Hsp70protein;. (b) a (C-terminal) fragment of (a) comprising the amino acidsequence TKDNNLLGRFELSG; (c) a (poly)peptide comprising the amino acidsequence TKDNNLLGRFELSG; or (d) a combination of (a), (b) and/or (c). 2.The method of claim 1, wherein the Hsp70 protein, the (C-terminal)fragment thereof, the (poly)peptide comprising the amino acid sequenceTKDNNLLGRFELSG, or the combination thereof is in an uncomplexed state.3. The method of claim 1 or 2, which is an in vivo method.
 4. The methodof claim 1 or 2, which is an ex vivo method.
 5. The method of claim 1 or2, which is an in vitro method.
 6. The method of claim 4 furthercomprising reinfusion of NK cells with induced or enhanced granzyme Bexpression into a mammal.
 7. The method of claim 6, wherein thereinfused NK cells are autologous and/or allogeneic NK cells.
 8. Themethod of claim 6 or 7, wherein said mammal is a human.
 9. The method ofany one of claims 1 to 8 wherein said contacting is effected for atleast 12 hours.
 10. The method of claim 9, wherein said contacting iseffected for at least 4 weeks.
 11. The method of any one of claim 1 to10 wherein said NK cells are prior to said contacting, obtained frombone marrow by incubating said bone marrow cells with interleukin-15(IL-15) and stem cell factor (SCF) at concentrations of 1 ng/ml-1000ng/ml per cytokine for at least 7 days up to 4 months.
 12. Use of NKcells which produce granzyme B after stimulation with (a) Hsp70 protein;(b) a (C-terminal) fragment of (a) comprising the amino acid sequenceTKDNNLLGRFELSG; (c) a (poly)peptide comprising the amino acid sequenceTKDNNLLGRFELSG; or (d) a combination of (a), (b) and/or (c); for thepreparation of a pharmaceutical composition for the treatment of tumors,viral or bacterial infections or inflammatory diseases.
 13. The useaccording to claim 12 wherein the NK cells are stimulated by a methodaccording to any of claims 1 to
 11. 14. Use of granzyme B for thepreparation of pharmaceutical composition for the perforin-independenttreatment of tumors, viral or bacterial infections or inflammatorydiseases.
 15. The use of claim 14 wherein granzyme B is used as the onlypharmaceutically active compound in said pharmaceutical composition. 16.A method of treating tumor, viral or bacterial infections orinflammatory diseases comprising of: (a) contacting NK-cells with tumorcells bearing Hsp70 on their surface or cells affected by said infectionor inflammation and bearing Hsp70 on their surface; (b) allowinggranzyme B to enter said cells via ion channels formed by said Hsp70 onthe cell surface; and (c) allowing said cells to undergo apoptosis as aresult of the enzymatic activity of granzyme B.
 17. A method treatingtumor, viral or bacterial infections or inflammatory diseases comprisingof: (a) contacting tumor cells bearing Hsp70 on their surface or cellsaffected by said infection or inflammation and bearing Hsp70 on theirsurface with granzyme B; a. allowing granzyme B to enter said cells viaion channels formed by said Hsp70 on the cell surface; and b. allowingsaid cells to undergo apoptosis as a result of the enzymatic activity ofgranzyme B.
 18. The method of claim 16 or 17, wherein granzyme B isadministered in a final concentration of 1 μg/ml to 500 μg/ml.
 19. Themethod of claim 16 or 17, wherein granzyme B is administered in a finalconcentration of 1 ng/ml to 10 ng/ml.
 20. The method of claim 19 whereingranzyme B is administered in a final concentration of about 6 ng/ml.21. The use of claim 14 or 15 or the method of any one of claims 16 to20 wherein granzyme B is packaged in liposomes.
 22. The use of any oneof claims 12 to 15 or 21 wherein said tumors comprise tumor cells whichexpress Hsp70 on the surface of their membrane or wherein cells affectedby said infection or inflammation express Hsp70 on the surface of theirmembrane.
 23. The use of any one of claims 12 to 15, 21 or 22 or themethod of any one of claims 16 to 22, wherein said tumors are selectedfrom a group consisting of stomach, gastric, colorectal, lung, pancreas,mammary, gynecological, head and neck tumors, dermatological tumors(e.g. melanoma), neuronal tumors, leukemia and lymphoma.
 24. The use ofany one of claims 12 to 15, 21 or 22 or the method of any one of claims16 to 22, wherein the viral infection is an infection by HIV orHepatitis virus.